Regulation of bcl-2 gene expression

ABSTRACT

The present invention provides novel anticode oligomers and methods of using them for controlling the growth of cancer cells expressing the bcl-2 gene.

RELATED APPLICATION DATA

This application is a continuation-in-Part of Ser. No. 07/840,716 filedFeb. 21, 1992, which was a continuation in part of Ser. No. 07,288,692filed Dec. 22, 1988, which has been abandoned.

REFERENCE TO GOVERNMENT GRANTS

The research in this patent application was supported in part byNational Institutes of Health grant CA 26380. The United Statesgovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of treatments for cancer andmore particularly to the field of anticode oligomer treatments forcancer.

BACKGROUND OF THE INVENTION

Current approaches to cancer treatment suffer from a lack ofspecificity. The majority of drugs that have been developed are naturalproducts or derivatives that either block enzyme pathways or randomlyinteract with DNA. Due to low therapeutic indices, most cancer treatmentdrugs are accompanied by serious dose-limiting toxicities. Theadministration of drugs to treat cancer kills not only cancer cells butalso normal non-cancerous cells. Because of these deleterious effects,treatments that are more specific for cancerous cells are needed.

It has been found that a class of genes, the oncogenes, plays a largerole in the transformation and maintenance of the cancerous state andthat turning off these genes, or otherwise inhibiting their effects, canreturn a cell to a normal phenotype. The role of oncogenes in theetiology of many human cancers has been reviewed in Bishop, “CellularOncogenes and Retroviruses,” Science, 235: 305-311 (1987). In many typesof human tumors, including lymphomas and leukemias, the human bcl-2 geneis overexpressed, and may be associated with tumorigenicity (Tsujimotoet al. Involvement of the bcl-2 gene in human follicular lymphoma,Science 228: 1440-1443 (1985)).

Antisense oligodeoxynucleotides are one example of a specifictherapeutic tool with the potential for ablating oncogene function.These short (usually about 30 bases) single-stranded synthetic DNAs havea complementary base sequence to the target mRNA and form a hybridduplex by hydrogen bonded base pairing. This hybridization can beexpected to prevent expression of the target mRNA code into its proteinproduct and thus preclude subsequent effects of the protein product.Because the mRNA sequence expressed by the gene is termed the sensesequence, the complementary sequence is termed the antisense sequence.Under some circumstances, inhibition of mRNA would be more efficientthan inhibition of an enzyme's active site, since one mRNA moleculegives rise to multiple protein copies.

Synthetic oligodeoxynucleotides complementary to (antisense) mRNA of thec-myc oncogene have been used to specifically inhibit production ofc-myc protein, thus arresting the growth of human leukemic cells invitro, Holt et al., Mol. Cell Biol. 8: 963-973 (1988), and Wickstrom etal., Proc. Natl. Acad. Sci. USA, 85: 1028-1-32 (1988).oligodeoxynucleotides have also been employed as specific inhibitors ofretroviruses, including the human immunodeficiency virus (HIV-I),Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA, 75: 280-284 (1978)and Zamecnik et al., Proc. Natl. Acad. Sci. USA, 83: 4143-4146 (1986).

SUMMARY OF THE INVENTION

The invention provides anticode oligomers and methods for inhibitinggrowth of cancer cells. The growth of lymphoma or leukemia cells, whichare types of lymphocytes, are inhibited by the anticode oligomers andmethods of the invention. An anticode oligomer complementary to at leastan effective portion of the mRNA sense strand to the human bcl-2 gene isprovided and cells are then contacted with the anticode oligomer in aconcentration sufficient to inhibit growth of the cells. The methods ofthe invention are suitable for inhibiting growth of lymphoma/leukemiacells that express the human bcl-2 gene and have a t (14; 18)chromosomal translocation as well as those that express the bcl-2 genebut do not have a t (14; 18) chromosomal translocation.

In accordance with preferred embodiments, the anticode oligomer issubstantially complementary to a strategic site in the pre-mRNA sensestrand or substantially complementary to the mRNA. A preferred strategicsite is the translation-initiation site of the pre-mRNA coding strand.Alternative strategic sites include coding sites for splicing, transportor degradation. The subject anticode oligomer either in its “native,”unmodified form—oligonucleotide—or as a derivative, is brought intocontact with the target lymphoma or leukemia cells. For in vivotherapeutic use, a derivative of the “native” oligonucleotide, such asthe phosphorothioate form is preferable since it is believed that theseforms are more resistant to degradation, notwithstanding the fact thatresponse times to some analogues, such as the phosphorothioate analogs,has been found to be somewhat slower than to the “native” form of theoligoynucleotide.

A preferred anticode oligomer, denominated herein the TI-AS (translationinitiation anticode oligomer) is an oligodeoxynucleotide which straddlesthe translation-initiation site of the mRNA coding strand of the humanbcl-2 gene and is complementary to this region. More preferably, thisnucleotide comprises a TAC portion which is complementary to the ATGinitiation sequence of the coding strand for the bcl-2 gene, andpreferably further comprises flanking portions of two to about onehundred bases, more preferably from about five to about twenty bases,which are complementary to portions of the bcl-2 gene coding strandflanking said initiation sequence. The TI-AS nucleotide has been foundeffective at inhibiting the growth of the target cells both in thepresence and absence of serum.

Alternatively, the anticode oligomer comprises an antisense nucleotidecomplementary to at least an effective portion of the splice donor siteof the pre-mRNA coding strand for the human bcl-2 gene. Moreparticularly, this nucleotide comprises a CA portion which iscomplementary to the GT splice donor of the bcl-2, and again comprisesflanking portions of two to about one hundred bases, preferably fromabout five to about twenty bases, which are complementary to portions ofthe bcl-2 gene coding strand flanking said splice donor.

In yet another embodiment, the anticode oligomer is complementary to atleast an effective portion of the splice acceptor region of the pre-mRNAcoding strand for the human bcl-2 gene. This oligomer comprises at leasta TC portion which is complementary to the AG splice acceptor of thebcl-2 gene, and again comprises flanking portions of two to about onehundred, preferably from about five to about twenty bases which arecomplementary to portions of the bcl-2 gene coding strand flanking saidacceptor. The subject oligomer may also be selected to overlap thecoding site for the 26 kDa protein, bcl-2-alpha or for the 22 kDaprotein, bcl-2-beta, protein products of the bcl-2 gene. Preferably theoligomer is selected to minimize homology with anticode oligomers forpre-mRNA or mRNA coding strands for other gene sequences.

Accordingly, a primary object of the present invention is the provisionof novel anticode oligomers, which are useful in inhibiting the growthof cancer cells. The present invention also includes compositions forinhibiting the growth of tumor cells, which compositions comprise theanticode oligomer of the present invention together with apharmaceutically acceptable carrier.

A further object of the present invention is the provision of methodsfor inhibiting the growth of cancer cells using said anticode oligomers.As a feature of the present invention, it was discovered that averagereductions of 30-40% in the relative levels of bcl-2 protein markedlyenhanced the sensitivity of lymphoma cells, in particular,t(14;18)-containing lymphoma cell lines to cancer chemotherapeuticagents, including conventional anticancer drugs. Such reductions wereachieved by introducing into tumor cells an anticode oligomer whichbinds to either pre-mRNA or mRNA expressed from the bcl-2 gene. Twomethods were used in the present invention to introduce said anticodeoligomers t: tumor cells. One method involved contacting the tumor cellswith a composition comprising the anticode oligomers. Another methodinvolved transfecting the tumor cells with a vector encoding anantisense oligonucleotide. Introducing an anticode oligomer to tumorcells achieved a reduction of bcl-2 expression and increases thechemosensitivity of neoplastic cells to cancer chemotherapeutic agentsor anticancer drugs.

Accordingly, the present invention achieved a method of killing tumorcells by introducing to tumor cells anticode oligomers which reducebcl-2 gene expression or impair Bcl-2 protein function before contactingthe cells with cancer chemotherapeutic agents. The cancerchemotherapeutic agents reduced the numbers of viable malignant cells,and the portion of tumor cells killed was greater than the portion whichwould have been killed by the same amount of drug in the absence ofintroducing the anticode oligomer oligodeoxynucleotide to the cells.

These and other objects of the present invention will become apparentfrom-the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs of the effects of varying concentrations ofantisense oligodeoxynucleotides on inhibition of cell proliferation.

FIG. 2 shows graphs of the concentration dependence of inhibition ofcell proliferation by antisense normal and phosphorothioateoligodeoxynucleotides. oligodeoxynucleotide additions to culturesincluded TI-AS phosphorothioate (o and ●; two separate experiments),TI-S phosphorothioate (▴), TI-AS normal (□), and TI-S normal (Δ).

FIG. 3 shows the results of gel electrophoresis of six antisenseoligonucleotides targeted against the translation initiation site ofbcl-2 mRNA.

FIG. 4 shows the degree of DNA fragmentation resulting fromoligonucleotide treatment of RS11846 cells. FIG. 4(a) shows the effectof oligonucleotides targeted against the translation initiation site.FIG. 4(b) shows the effect of oligonucleotides directed against. the5′-cap region of bcl-2 mRNA.

FIG. 5 is a graph showing the concentration—dependence of inhibition byan antisense oligonucleotide targeted against the translation initiationsite of bcl-2 mRNA.

FIGS. 6(a) and (b) are graphs showing the results of immunofluorescenceanalysis of bcl-2 protein levels in oligonucleotide-treated cells.

FIGS. 7(a)-(d) are FACS profiles for 697 cells before and aftertreatment with bcl-2 antisense oligonucleotides.

FIG. 8(a)-(c) show bcl-2 antisense oligodeoxynucleotides producingsequence-specific reductions in bcl-2 mRNA and bcl-2 protein andproducing increased sensitivity of SU-DHL-4 cells to cancerchemotherapeutic drugs.

FIG. 9 demonstrates the differential effects of bcl-2 antisenseoligomers on chemosensitivity of 32D-bcl-2 and 32D-BHRF-1 cells.

FIG. 10(a-b) shows reduction of chemoresistance of RS11846 cells frominducible bcl-2 antisense expression from an expression plasmid.

FIG. 11 shows methylphosphonate/phosphodiester bci-2 antisense oligomersinducing death of DOHH2 lymphoma cells.

FIG. 12 shows methylphosphonate (MP)/Phosphodiester (PO) chimericoligomers inhibiting growth of MCF-7 human breast cancer cells.

FIG. 13 shows optimization of antisense bcl-2 oligomer sequences.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, anticode oligomers are provided forinhibiting cancer cell growth, for increasing the sensitivity of cancercells to cancer chemotherapeutic agents, or for inducing cancer celldeath alone or in combination with any one or more cancerchemotherapeutic agents.

Definitions

As used herein, the term “anticode oligomers” means anticodeoligonucleotides and analogs thereof and refers to a range of chemicalspecies that recognize polynucleotide target sequences through hydrogenbonding interactions with the nucleotide bases of the target sequences.The target sequences may be single- or double-stranded RNA or single- ordouble-stranded DNA.

The anticode oligonucleotides and analogs thereof may be RNA or DNA, oranalogs of RNA or DNA, commonly referred to as antisense oligomers orantisense oligonucleotides. Such RNA or DNA analogs comprise but are notlimited to 2-′O-alkyl sugar modifications, methylphosphonate,phosphorothiate, phosphordithioate, formacetal, 3′-thioformacetal,sulfone, sulfamate, and nitroxide backbone modifications, and analogswherein the base moieties have been modified. In addition, analogs ofoligomers may be polymers in which the sugar moiety has been modified orreplaced by another suitable moiety, resulting in polymers whichinclude, but are not limited to, morpholino analogs and peptide nucleicacid (PNA) analogs (Egholm, et al. Peptide Nucleic Acids(PNA)—oligonucleotide Analogues with an Achiral Peptide Backbone,(1992)).

Anticode analogs may also be mixtures of any of the oligonucleotideanalog types together or in combination with native DNA or RNA. At thesame time, the oligonucleoltides and analogs thereof may be used aloneor in combination with one or more additional oliognucleotides oranalogs thereof. The oligonucleotides may be from about 10 to about1,000 nucleotides long. Although oliognucleotides of 10 to 100nucleotides are useful in the invention, preferred oligonucleotidesrange from about 15 to about 24 bases in length.

Anticode oligonucleotides and analogs thereof also comprise conjugatesof the oligonucleotides and analogs thereof. (John Goodchild, Congugatesof Oligonucleotides and Modified Oligonucleotides: A Review of TheirSynthesis and Properties, Bioconjugate Chemistry, Volume 1 No. 3,May/June (1990)). Such conjugates having properties to improve theuptake, pharmacokinetics, and nuclease resistance of theoligonucleotide, or the ability to enhance cross-linking or cleavage ofthe target sequence by the oligonucleotide.

As used herein, the term “cell proliferation” refers to cell divisionrate/cell cycle. The term “growth,” as used herein, encompasses bothincreased cell numbers due to faster cell division and due to slowerrates of cell death.

As used herein, bcl-2 gene expression refers to bcl-2 protein productionfrom the human bcl-2 gene; e.g. reduced bcl-2 gene expression meansreduced levels of bcl-2 protein.

As used herein, “strategic sites” are defined as any site which whenbound by the claimed anticode molecules or analogs thereof results ininhibiting expression of the bcl-2 gene.

As used herein, the term “sequence portion” is a portion of thenucleotide sequence of an RNA oligonucleotide. In appropriate contexts,“sequence portion” may refer to a portion of the nucleotide sequence ofa DNA segment or DNA oligonucleotide.

Uncontrolled cell proliferation is a marker for a cancerous or abnormalcell type. Normal, non-cancerous cells divide regularly, at a frequencycharacteristic for the particular type of cell. When a cell has beentransformed into a cancerous state, the cell divides and proliferatesuncontrollably. Inhibition of proliferation modulates the uncontrolleddivision of the cell. Containment of cell division often correlates witha return to a non-cancerous state.

A human gene termed bcl-2 (B cell lymphoma/leukemia-2) is implicated inthe etiology of some common lymphoid tumors, Croce et al., “MolecularBasis Of Human B and T Cell Neoplasia,” in: Advance i. Viral Oncology,7: 35-51, G. Klein (ed.), New York: Raven Press, 1987. High levels ofexpression of the human bcl-2 gene have been found in all lymphomas witht (14; 18) chromosomal translocations including most follicular B celllymphomas and many large cell non-Hodgkin's lymphomas. High levels ofexpression of the bcl-2 gene have also been found in certain leukemiasthat do not have a t(14; 18) chromosomal translocation, including mostcases of chronic lymphocytic leukemia acute, many lymphocytic leukemiasof the pre-B cell type, neuroblastomas, nasophryngeal carcinomas, andmany adenocarcinomas of the prostate, breast, and colon. (Reed et al.,Differential expression of bcl-2 protooncogene in neuroblastoma andother human tumor cell lines of neural origin. Cancer Res. 51:6529(1991); Yunis et al. Bcl-2 and other genomic alterations in theprognosis of large-cell lymphomas. New England J. Med. 320:1047; Camposet al. High expression of bcl-2 protein in acute myeloid leukemia isassociated with poor response to chemotherapy. Blood 81:3091-3096(1993); McDonnell et al. Expression of the protooncogene bcl-2 and itsassociation with emergence of androgen-independent prostate cancer.Cancer Res. 52:6940-6944 (1992); Lu Q-L, et al. Bcl-2 protooncogeneexpression in Epstein Barr Virus-Associated Nasopharyngeal Carcinoma,Int. J. Cancer 53:29-35 (1993); Bonner et al. bcl-2 protooncogene andthe gastrointestinal mucosal epithelial tumor progression model asrelated to proposed morphologic and molecular sequences, Lab Invest.68:43A (1993)).

While not limited to the following explanation, the present inventionexploits cellular mechanisms concerned with normal cell death. Becausemost types of cells have a finite life span and are programmed to die,uncontrollable cell accumulation can also result because of a defect innormal cell death mechanisms rather than through an increased rate ofcell division. The bcl-2 gene contributes to the pathogenesis of cancerprimarily by prolonging cell survival rather than accelerating celldivision.

Antisense oligomers suitable for use in the invention include nucleotideoligomers which are two to two hundred nucleotide bases long; morepreferably ten to forty bases long; most preferably twenty bases long.The oligonucleotides are preferably selected from those oligonucleotidescomplementary to strategic sites along the pre-mRNA of bcl-2, such asthe translation initiation site, donor and splicing sites, or sites fortransportation or degradation.

Blocking translation at such strategic sites prevents formation of afunctional bcl-2 gene product. It should be appreciated, however, thatany combination or subcombination of anticode oligomers, includingoliognucleotides complementary or substantially complementary to thebcl-2 pre-mRNA or mRNA that inhibit cell proliferation is suitable foruse in the invention. For example, oligodeoxynucleotides complementaryto sequence portions of contiguous or non-contiguous stretches of thebcl-2 RNA may inhibit cell proliferation and would thus be suitable foruse in the invention.

It should also be appreciated that anticode oligomers suitable for usein the invention may also include oligonucleotides flanking thosecomplementary or substantially complementary to such sequence portionsas the strategic or other sites along the bcl-2 mRNA. The flankingsequence portions are preferably from two to about one hundred bases,more preferably from about five to about twenty bases in length. It isalso preferable that the anticode oligomers be complementary to asequence portion of the pre-mRNA or mRNA that is not commonly found inpre-RNA or mRNA of other-genes to minimize homology of anticodeoligomers for pre-mRNA or mRNA coding strands from other genes.

Preferred antisense, or complementary, oligodeoxynucleotides are listedin Table 1. TABLE I bcl-2 Oligodeoxynucleotides translation initiationantisense (TI-AS) 3′...CCCTTCCTACCGCGTGCGAC...5′ bcl-25′...CTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGA...3′ splice donor antisense(SD-AS) 31...CCTCCGACCCATCCACGTAG...5′ bcl-25′...ACGGGGTAC...GGAGGCTGGGTAGGTGCATCTGGT...3′ splice acceptor antisense(SA-AS) 3′...GTTGACGTCCTACGGAAACA...5′ bcl-25′...CCCCCAACTGCAGGATGCCTTTGTGGAACTGTACGG...3′

It will be appreciated by those skilled in the art to which thisinvention pertains, that anticode oligomers having a greater or lessernumber of substituent nucleotides, or that extend further along thebcl-2 mRNA in either the 3′ or 5′ direction than the preferredembodiments, but which also inhibit cell proliferation are also withinthe scope of the invention.

It is preferable to use chemically modified derivatives or analogs ofanticode oligomers in the performance of the invention rather than“native” or unmodified oligodeoxynucleotides. “Native”oligodeoxynucleotides can be conveniently synthesized with a DNAsynthesizer using standard phosphoramidite chemistry. Suitablederivatives, and methods for preparing the derivatives, includephosphorothioate, Stein et al., Nucl. Acids Res., 16:3209-3221 (1988);methylphosphonate, Blake et al., Biochemistry 24:6132-6138 (1985) andalphadeoxynucleotides, Morvan et al., Nucl. Acids Res. 14:5019-5032(1986), 2′-O-methyl-ribonucleosides (Monia et al. Evaluation of2′-modified oligonucleotides containing 2′ deoxy gaps as antisenseinhibitors of gene expresssion. J. Biol. Chem. 268:14514-14522 (1933)),and covalently-linked derivatives such as acridine, Asseline et al.,Proc. Natl. Acad. Sci. USA 81:3297-3201 (1984); alkylated (e.g.,N-2-chlorocethylamine), Knorre et al., Biochemie 67:783-789 (1985) andVlassov et al., Nucl. Acids Res. 14:4065-4076 (1986); phenazine, Knorreet al., supra, and Vlassov et al., supra;5-methyl-N⁴-⁴-N-ethanocytosine, Webb et al., Nucl. Acids Res.14:7661-7674 (1986); Fe-ethylenediamine tetraacetic acid (EDTA) andanalogues, Boutorin et al., FEBS Letter's 172:43-46 (1984);5-glycylamido-1, 10-o-phenanthroline, Chi-Hong et al., Proc. Natl. Acad.Sci. USA 83: 7147-7151 (1986); and diethylenetriaamine-pentaacetic acid(DTPA) derivatives, Chu et al., Proc. Natl. Acad. Sci. 82: 963-967(1985). All of the above publications are hereby specificallyincorporated by reference as if fully set forth herein.

The anticode oligomer of the present invention can also be combined witha pharmaceutically acceptable carrier for administration to a subject orfor ex-vivo administration. Examples of suitable pharmaceutical carriersare a variety of cationic lipids, including, but not limited toN-(1-2,3-dioleyloxy)propyl)-n,n,n-trimethylammonium chloride (DOTMA) anddioleoylphophotidylethanolamine (DOPE)]. Liposomes are also suitablecarriers for the anticode oligomers of the invention.

The anticode oligomers may be administered to patients by any effectiveroute, including intravenous, intramuscular, intrathecal, intranasal,intraperitoneal, subcutaneous injection, in situ injection and oraladministration. Oral administration requires enteric coatings to protectthe claimed anticode molecules and analogs thereof from degradationalong the gastrointestinal tract. The anticode oligomers may be mixedwith an amount of a physiologically acceptable carrier or diluent, suchas a saline solution or other suitable liquid. The anticode oligomersmay also be combined with liposomes or other carrier means to protectthe anticode molecules or analogs thereof from degradation until theyreach their targets and/or facilitate movement of the anticode moleculesor analogs thereof across tissue barriers.

The anticode oligomers may also be useful for ex vivo bone marrowpurging. Normally, the amounts of conventional cancer chemotherapeuticagents or drugs and irradiation that a patient can receive are limitedby toxicity to the marrow, i.e., anemia (fatigue, heart failure),thrombocytopenia (bleeding), neutropenia (infection). Thus, in order todeliver sufficient concentrations of drugs and irradiation to totallyeradicate the tumor, the physician would simultaneously destroy thepatient's normal bone marrow cells leading to patient demise.Alternatively, large amounts of bone marrow can be surgically extractedfrom the patient and stored in vitro. while the patient receivesaggressive conventional treatment. The patient can then be rescued byreinfusion of their own bone marrow cells, but only if that marrow hasbeen “purged” of residual malignant cells. The claimed anticodeoligomers could be used to remove residual malignant cells from the bonemarrow.

The anticode oligomers are administered in amounts effective to inhibitcancer or neoplastic cell growth. The actual amount of any particularanticode oligomer administered will depend on factors such as the typeof cancer, the toxicity of the anticode oligomer to other cells of thebody, its rate of uptake by cancer cells, and the weight and age of theindividual to whom the anticode oligomer is administered. Because ofinhibitors present in human serum that may interfere with the action ofthe anticode oligomer an effective amount of the anticode oligomer foreach individual may vary. An effective dosage for the patient can beascertained by conventional methods such as incrementally increasing thedosage of the anticode oligomer from an amount ineffective to inhibitcell proliferation to an effective amount. It is expected thatconcentrations presented to cancer cells in the range of about 0.001micromolar to about 100 micromolar will be effective to inhibit cellproliferation.

The anticode oligomers are administered to the patient for at least atime sufficient to inhibit proliferation of the cancer cells. Theanticode oligomers are preferably administered to patients at afrequency sufficient to maintain the level of anticode oligomers at aneffective level in or around the cancer cells. To maintain an effectivelevel, it may be necessary to administer the anticode oligomers severaltimes a day, daily or at less frequent intervals. Anticode oligomers areadministered until cancer cells can no longer be detected, or have beenreduced in number such that further treatment provides no significantreduction in number, or the cells have been reduced to a numbermanageable by surgery or other treatments. The length of time that theanticode oligomers are administered will depend on factors such as therate of uptake of the particular oligodeoxynucleotide by cancer cellsand time needed for the cells to respond to the oligodeoxynucleotide. Invitro, maximal inhibition of neoplastic cell growth by “native,”unmodified anticode oligomers occurred two days after initiation ofcultures,, whereas phosphorothioate oligodeoxynucleotides required 4 to7 days to achieve maximal inhibition. In vivo, the time necessary formaximal inhibition of cell proliferation may be shorter or longer.

The anticode oligomers of the invention may be administered to patientsas a combination of two or more different anticode oligomeroligodeoxynucleotide sequences or as a single type of sequence. Forinstance, TI-AS and SD-AS could be administered to a patient or TI-ASalone.

It is also believed that the anticode oligomers of the invention may beuseful in the treatment of autoimmune diseases. Autoimmune diseases arethose diseases in which the body's immune system has malfunctioned insome way. Administration of the anticode oligomers of the invention to aperson having an autoimmune disease should inhibit proliferation ofbcl-2 overexpressing lymphocytes, which would in turn reduce thesymptoms of the autoimmune disease. For use in treating autoimmunediseases, the anticode oligomers would be administered as describedherein.

EXAMPLES

General Methods

The Examples below use the following protocols:

A. Cells and Cell Cultures. Hunan leukemic cells lines used for thesestudies were RS11846 follicular lymphoma cells, 697 pre-B cell acutelymphocytic leukemic cells, and JURAT T cell acute lymphocytic leukemiccells as described in Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83:5214-5218 (1986) and Weiss et al., Proc. Natl. Acad. Sci. US.,138:2169-2174 (1987). Human peripheral blood lymphocytes (PBL) wereisolated from fresh whole blood as described in Reed et al., J.Immunol., 134:314-319 (1985). All lymphoid cells were cultured at 5×10⁵cells/ml in RPMI medium supplemented with 1 mM glutamine, antibiotics,and either 5-10% (v:v) fetal bovine serum (FBS), 5-10% (v:v) calf serum(CS) (both from Hyclone Laboratories), or 1% (v:v) HLI concentratedsupplement (Ventrex Laboratories) for serum-free cultures. Murinefibroblast cell lines were added at 10³ cells/cm² in DMEM mediumcontaining glutamine, antibiotics and 5-10% (v:v) FCS. Fibroblast celllines were NIH 3T3 cells, 3T3-B-alpha-S cells, and 3T3-B-alpha-AS cells.These latter two cell lines are NIH 3T3 cells that express high levelsof a human bcl-2-alpha cDNA in either the sense or antisenseorientation, respectively, by virtue of stable transfection withexpression vectors constructs.

B. Measurement of Cellular Growth. Growth of cell lines cultured in thepresence or absence of anticode oligomers was measured by two methods:cell counts using a hemocytometer; and DNA synthesis by assaying[³]-thymidine incorporation essentially as described in Reed et al., J.Immunol., 134:314-319 (1985). Briefly, cells were cultured in 96-wellflat-bottomed microtiter plates (Falcon) at 0.2 ml/well. At appropriatetimes, cells were resuspended, 25 μl removed from cultures for cellcounting, and this volume replaced with 25 μl of 20 UCi/ML[³H]-thymidine (specific activity 6.7 Ci/mmole) (New England Nuclear).Microtiter cultures were then returned to 37° C. and 95% air: 5% CO₂atmosphere for 8 hours before lysing cells an glass filters anddetermining relative levels of [³H]-thymidine incorporation into DNA byscintillation counting. Cell counts were performed in the presence oftrypan blue dye to determine the concentration of viable cells induplicate microcultures.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] dyereduction assays were performed by the method of Tada, et al. J. ImmunolMethods 93, 1-7 (1986), and confirmed to be within the linear range ofthe assay under the conditions described here. The number of viablecells per well was extrapolated from standard curves that were includedwith each assay and that consisted of serial two-fold dilutions-ofexponentially growing SU-DHL-4 cells in HL-1 medium, beginning with 10⁶cells/ml (0.2 ml/well). Samples were assayed in triplicate and theOD600_(nm) for a media/reagent blank was subtracted from all valuesprior to calculations.

C. RNA Blot Analysis. Total cellular RNA was isolated by a quanidiniumisothiocyanate/phenol procedure as described in Chomczynski et al.,Analyt. Biochem., 162:156-139 (1987). The polyadenylated fraction waspurified by oligodeoxythymidine-cellulose chromatography as described inAviv et al., Proc. Natl. Acad. Sci. USA, 69:1408-1412 (1972).Approximately 5 μg aliquots of mRNA were size-fractionated in 0.8%agarose/6% formaldehyde gels and transferred to nylon membranes. Blotswere prehybridized, hybridized, and washed exactly as described in Reedet al., Mol. Cell Biol., 5:3361-3366 (1985), using either a ³²P-cDNA forhuman bcl-2, as described in Tsujimoto et al., Proc. Natl. Acad. Sci.USA, 83:5214-5218 (1986), or a murine bcl-2 probe, pMBCL5.4 as describedin Negrini et al., Cell, 49:455-463 (1987). Blots were exposed to KodakXAR film with intensifying screens at −70° C. for 1-10 days. fluting³²P-bcl-2 probes from membranes and rehybridizing with a ³²P probe formouse beta-2-microglobulin verified nearly equivalent amounts of mRNAfor all samples on blots.

EXAMPLE 1 Preparation of Anticode Oligomers

Normal and phosphorothioate oligodeoxynucleotides were synthesized usingan Applied Biosystems 380B DNA synthesizer, and purified by HPLCreverse-phase chromatography (PRP-1 column) as described in Stein etal., Nucl. Acids Res., 16:3209-3221 (1988) which is specificallyincorporated as if fully set forth herein. In some cases it wasnecessary to further purify oligodeoxynucleotides by C18-Sep-Pakchromatography (Waters Associates, Millipore, Inc.), as describedpreviously in Kern et al., J. Clin. Invest., 81:237-244 (1988), toeliminate nonspecific cytotoxic activity. oligodeoxynucleotides elutedin 30% acetonitrile were evaporated to dryness, resuspended at 1-2 mM insterile Dulbecco's phosphate-buffered saline or Hanks' buffered saltsolution (both from Gibco), and stored at −80 C in small aliquots.

Table 1 shows the oligodeoxynucleotides synthesized and their relationto the sense-strand of the human bcl-2 gene. Portions of the sequence ofthe coding strand of the human bcl-2 gene are shown, including thetranslation initiation site (top), splice donor site (middle), spliceacceptor region (bottom), and emperically selected sites within the 5′untranslated portion of bcl-2 pre-mRNA. The ATG initiation codon, GTsplice donor, and AG splice acceptor consensus sequences are in boxes.

The sequences of the oligodeoxynucleotides synthesized for theseinvestigations are presented, and their relation to human bcl-2 mRNA isindicated. The TI-AS oligodeoxynucleotide is antisense at thetranslation initiation site and TI-S is its complementary sense version.SD-AS and SD-S are oligodeoxynucleotides having antisense and senseorientations, respectively, relative to the splice donor region.

The oligodeoxynucleotide TI-AS straddles the predictedtranslation-initiation site of bcl-2 mRNAs and is complementary(antisense) to this region. As a control, the sense version of this 20bp oligodeoxynucleotide, TI-S, was also synthesized.

In an effort, to specifically block splicing of bcl-2 mRNAs, a 20 bpantisense oligodeoxynucleotide, SD-AS, was synthesized that overlaps thesplice donor site in bcl-2 primary transcripts. In addition, acomplementary sense oligodeoxynucleotide, SD-S, was prepared as depictedin Table 1. The human bcl-2 gene gives rise to several transcriptsthrough alternative splice site selections, see Tsujimoto et al., Proc.Natl. Acad. Sci. USA, 83:5214-5218 (1986). The preponderance of thesetranscripts depend upon splicing and encode a 26 kDa protein,bcl-2-alpha. One minor transcript, however, does not undergo a spliceand consequently encodes a 22 kDa protein bcl-2-beta. The SD-ASoligodeoxynucleotide can thus potentially block maturation of most butnot all bcl-2 transcripts.

EXAMPLE 2 Treatment of Serum for In Vitro Investigations of AntisenseNormal Oligodeoxynucleotides

Because normal oligodeoxynucleotides are sensitive to degradation bynucleases present in serum, the efficacy of the TI-ASoligodeoxynucleotide in fetal bovine serum (FBS) heated for 30 minutesat 56° c. (the usual procedure for inactivating serum complement) wascontrasted with the efficacy of TI-AS in FBS heated for 1 hour at 68°C., a temperature sufficient for irreversible inactivation of manynucleases. The RS11846 follicular lymphoma cell line was used. RS11846cells contain a t (14; 18) chromosomal translocation that deregulatesbcl-2 expression, resulting in the accumulation of high levels of bcl-2mRNAs, Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83: 5214-5218(1986).

RS11846 follicular lymphoma cells were cultured in medium containing 5%(vol:vol) fetal bovine serum (FBS) that had been heated at 56° C. for0.5 hours or at 68° C. for 1 hour. TI-AS normal oligodeoxynucleotide wasadded at the initiation of culture, and the density of viable cellsdetermined two days later.

The TI-AS normal oligodeoxynucleotide was more effective in 68 C-treatedserum at suppressing the growth in culture of these lymphoma cells. Inall subsequent experiments, sera heated at 68° C. for 1 hour prior touse were used in cultures. This treatment did not impair thegrowth-supporting capacity of the sera.

EXAMPLE 3 Specific Inhibition of Lymphoid Cell Growth by AntisenseNormal Oligodeoxynucleotides

Antisense normal oligodeoxynucleotides directed against the translationinitiation site (TI-AS) and the splice donor site (SD-AS) of bcl-2transcripts were tested for their ability to suppress the proliferationof normal and neoplastic lymphoid cell's.

RS11846 follicular lymphoma cells, 7-JUKRAT T cell leukemia cells, andfreshly isolated peripheral blood lymphocytes were cultures in mediumcontaining 10% (vol:vol) FBS that had been heated at 68° C. for one hourvarious concentrations of normal oligodeoxynucleotides were added at theinitiation of culture, including: TI-AS, TI-S, SD-AS, and SD-S. RelativeDNA synthesis was measured in cultures after 2-3 days by [³H]-thymidineincorporation. Data were calculated as a percentage of control culturescontaining volumes of PBS or HBSS equivalent tooligodeoxynucleotide-treated cultures, and represent the mean (±standarddeviation) of duplicate cultures.

Similar data were obtained by measuring cell counts, excluding coldthymidine inhibition as an explanation for the suppression of DNAsynthesis observed in cultures treated with antisenseoligodeoxynucleotides.

As shown in FIG. 1, both the TI-AS and SD-AS oligodeoxynucleotidesinhibited the growth of RS11846 cells in a concentration-dependentmanner. The SD-AS oligonucleotide was less effective in inhibiting cellgrowth than the TI-AS oligodeoxynucleotide. In contrast to theseantisense oligodeoxynucleotides, sense oligodeoxynucleotides (TI-S andSD-S) were not inhibitory even at concentrations of up to 250 μG/ml.Moreover, non-sense oligodeoxynucleotides (i.e., those having the samebase composition as the antisense oligodeoxynucleotides but withscrambled sequences) also failed to suppress the proliferation ofRS11846 cells. The data thus indicate that antisenseoligodeoxynucleotides can specifically block the proliferation of thesetumor cells. Several other leukemic cell lines that express the bcl-2gene were also tested for inhibition of their proliferation by TI-AS andSD-AS oligonucleotides. As with the JURKAT T cell acute lymphocyticleukemic cells, in every case a specific and concentration-dependentdecrease in the growth of these human leukemic cells in culturescontaining antisense oligodeoxynucleotides was observed.

It has been demonstrated that bcl-2 expression is transiently induced innormal human peripheral blood lymphocytes (PBL) when these cells arestimulated to proliferate, suggesting that this gene may play a role inthe regulation of normal lymphocyte growth, Reed et al., Science 236:1295-1297 (1987). The capacity of antisense oligodeoxynucleotides toimpair the growth of PBL cultured with a monoclonal antibody, OKT3 (Vanden Elsen et al., Nature 312: 413-418 (1984)), that stimulates theirproliferation was therefore tested. PBL were stimulated with 50 ml ofpurified OKT3 monoclonal antibody. As shown in FIG. 1, the TI-ASoligodeoxynucleotide specifically suppressed the proliferation of PBL ina concentration-dependent manner. These antisense normaloligodeoxynucleotides thus suppressed the growth in culture of leukemiccells that constitutively express the bcl-2 gene and of normallymphocytes where in bcl-2 expression is inducible.

EXAMPLE 4 Time-Course of Inhibition by Antisense NormalOligodeoxynucleotides

The kinetics of inhibition by antisense oligodeoxynucleotides wasexamined in cultures of RS11846 follicular lymphoma cells and of 697pre-B cell acute lymphocytic leukemia cells. Both of these neoplastic Bcell lines transcribe and accumulate bcl-2 mRNAs at high levels,Tsujimoto et al., Proc. Natl. Acad. Sci. USA, 83:5214-5218 (1986).

RS11846 follicular lymphoma and 697 pre-B cell leukemia cells werecultured in medium containing 10% (vol:vol) 68° C.-treated FBS andnormal oligodeoxynucleotides. Cells were cultured with 50 μg/ml TI-AS,100 μg/ml SD-AS, 50 μg/ml TI-S(RS11846 cells) or 100 μg/ml SO-S (697cells), or PBS as a control. DNA synthesis (kcpm/10⁵ viable cells) andcell densities (10⁵ viable cells/ml) were measured at various timesafter initiation of cultures.

Antisense normal oligodeoxynucleotides markedly inhibited DNA synthesismeasured in cultures of these cells within 24 hours. Diminished celldensities were readily apparent in these cultures within 2 days.Antisense normal oligodeoxynucleotides thus rapidly inhibited the invitro growth of leukemic cells. The action of antisenseoligodeoxynucleotides was specific, since sense oligodeoxynucleotidesdid not impair proliferation in these cultures. Though cell viabilitiesoften declined during the later days of culture no increase in celldeath was seen during the first 1-2 days of culture with antisenseoligodeoxynucleotides, suggesting a non-cytotoxic mechanism.

EXAMPLE 5 Comparision of Different Serum Preparations

Inhibition of proliferation of leukemic cells with antisenseoligodeoxynucleotides can vary greatly depending on the lot of serumused in cultures.

To determine the effects of serum of inhibition of proliferation,relative levels of DNA synthesis were measured in cultures of 697 pre-Bcell leukemia cells 2 days after addition of 200 μM TI-AS normaloligodeoxynucleotide. Cells were cultured in medium supplemented with 1%(vol:vol) HL1-concentrate (serum-free condition), 5% (vol:vol) of twodifferent lots of calf serum (CS1 and CS2), or 5% (vol:vol) of twodifferent lots of fetal bovine serum (FBS1 and FBS2). All sera wereheated at 68° C. for 1 hour prior to use in cultures.

The normal TI-AS oligodeoxynucleotide markedly inhibited DNA synthesis(92%) and cellular proliferation in serum-free cultures (HL1) of 697cells. This antisense oligodeoxynucleotide was equally effective (94%)in cultures containing 5% (v:v) of one of the lots of fetal bovine serum(FBS2). In contrast, inhibition was significantly reduced in culturescontaining other serum preparations (CS1, CS2, FBS1). It has beengenerally observed that antisense normal oligodeoxynucleotides are lesseffective in cultures supplemented with calf serum (CS) than in thosecontaining fetal bovine serum (FBS).

EXAMPLE 6 Concentration Dependence of Inhibition by Antisense NormalOligodeoxynucleotides in Serum-Free Cultures

697 pre-B cell leukemia cells were cultured in medium with either 1%(vol:vol) HL1-concentrate (serum-free conditions or 5% (vol:vol) 68°C.-treated FBS2). Relative levels of DNA synthesis and cellulardensities measured after 2 days in cultures containing variousconcentrations of normal TI-AS oligodeoxynucleotide.

The TI-AS oligodeoxynucleotide was inhibitory at lower concentrationswhen used in serum-free cultures. At 100 μM, for instance, no inhibitionof cellular proliferation was seen in FBS2-containing cultures, whereascell counts were reduced by approximately 75% in serum-free cultures. Athigher concentrations of antisense oligodeoxynucleotides (200-250 μM),however, inhibition of 697 cellular proliferation was comparable in bothtypes of cultures. The increased efficacy of normaloligodeoxynucleotides in serum-free cultures was specific, since thesense oligonucleotide (TI-S) was not inhibitory at the sameconcentrations.

EXAMPLE 7 Antisense Phosphorothioate Oligodeoxynucleotides: Time Courseof Inhibition

To contrast the efficacy of phosphorothioate oligodeoxynucleotides withthat of normal oligodeoxynucleotides with regard to inhibition of humanleukemic cell growth, phosphorothioate oligodeoxynucleotides werecultured with 697 pre-B cell leukemia cells and the effects oninhibition were measured. 697 pre-B cell leukemia cells were cultured inserum-free medium for various times before measuring DNA synthesis(kcpm) and cell densities (10⁶ cells/ml). Cells were seeded at aninitial density of either 0.2×10⁵ cells/ml or 0.5×10⁵ cells/ml. Cultureconditions were 25 μM TI-AS phosphorathioate, 25 μM TI-Sphosphorothioate, and control cultures treated with HBSS.

To avoid experimental variation due to differences among lots of sera,697 leukemic cells were cultured in serum-free conditions. When culturedat an initial seeding density of 0.5×10⁶ cells/ml, 697 cells achievedmaximal DNA synthesis and cellular densities at 4-5 days. Addition of 25μM sense phosphorothioate oligodeoxynucleotide (TI-S) at the initiationof these cultures had little effect on 697 cell growth. In replicatecultures containing 25 μM antisense phosphorothioate (TI-AS), however,some diminution in DNA synthesis was evident within 2 days and wasmaximal at 4-5 days. Maximal inhibition of 697 cell growth, asdetermined by cell counts, was seen at 6 days after initiation ofcultures.

When 697 cells were initially seeded at 0.2×10⁶ cells/ml, the antisensephosphorothioate oligodeoxynucleotide, TI-AS, resulted in only slightinhibition at 2 days, attaining maximal suppression of DNA synthesis inthese cultures at day 7. As with normal oligodeoxynucleotides, thisinhibition by phosphorothioate oligodeoxynucleotides appeared to bemediated through non-cytotoxic mechanisms, since cellular viabilitiesdid not decline until late in the course of culture. Compared withnormal antisense oligodeoxynucleotides, therefore, phosphorothioateoligodeoxynucleotides had a slower onset of action.

EXAMPLE 8 Concentration Dependence of Inhibition by Antisense bcl-2Phosphorothioate Oligodeoxynucleotides

The concentration descendence of inhibition by phosphorothioate andnormal TI-AS oligodeoxynucleotides in cultures of 697 cells inserum-free medium was compared as follows.

697 cells were cultured in serum-free medium for either 3 days (normaloligodeoxynucleotides) or 4 days (phosphorothioateoligodeoxynucleotides) prior to measuring cell densities and levels ofDNA synthesis. Oligodeoxynucleotide additions to cultures included TI-ASphosphorothioate, TI-S phosphorothioate, TI-AS normal, and TI-S normal.

As shown in FIG. 2, TI-AS phosphorothioate oligodeoxynucleotidesmarkedly inhibited the proliferation of 697 cells at 25-50 μM. Incontrast, normal TI-AS oligodeoxynucleotides required concentrations 5-to 10-fold higher (approximately 250 μM) to cause a comparablesuppression of 697 cellular proliferation. Suppression by the antisensephosphorothioate oligodeoxynucleotide TI-AS was specific over thisconcentration range, since its complementary sense oligodeoxynucleotide(TI-S) produced little inhibition of 697 cell growth in replicatecultures (see FIG. 2).

EXAMPLE 9 Influence of Serum Preparation on Inhibition by AntisensePhosphorothioate Oligodeoxynucleotides

To further define the effects of serum preparation on the inhibitoryactivity of phosphorothioate oligodeoxynucleotides, FBS that had beenheated to 56° C. for 30 minutes, 68° C. for 1 hour, or not heated priorto addition to cultures was added to cultures of RS11846 lymphoma cells.

RS11846 cells were cultured in medium containing 1% (vol:vol)HL1-concentrate or 5% (vol:vol) FBS that had been heated at 56° C. for0.5 hour, 68° C. for 1 hour, or that had not been heated. Cell countswere calculated as a percentage relative to control cultures treatedwith equivalent concentrations of TI-S phosphorothioateoligodeoxynucleotide, and represent the mean percentage (standarddeviation was less than 10% for all values) for duplicate culturescounted on days 4 and 5.

The TI-AS phosphorothioate oligodeoxynucleotide completely inhibited thegrowth of RS11846 cells at 25 μM, with an estimated half-maximalinhibitory concentration of approximately 11 μM. In contrast, thisphosphorothioate oligodeoxynucleotide was considerably less effective incultures containing 5% (v:v) FBS. Furthermore, heating FBS prior toadding it to cultures did not significantly improve the ability of theTI-AS phosphorothioate oligodeoxynucleotide to suppress the growth ofRS11846 lymphoma cells. At an oligodeoxynucleotide concentration of 50μM, inhibition of proliferation of RS11846 cells never exceeded 48%serum-containing cultures, regardless of the heating procedure used.

EXAMPLE 10 Influence of Dialysis of Serum on Inhibition by Normal andPhosphorothioate Antisense Oligodeoxynucleotides

To further characterize the nature of the interfering substances inserum, experiments were performed wherein 68° C.-heated serum wasextensively dialyzed (molecular weight cutoff=3500) prior to being addedto cultures of 697 leukemic cells. Experiments were conducted with 12.5μM TI-AS phosphorothioate oligodeoxynucleotide and 200 μM of the normaloxygen-based TI-AS oligodeoxynucleotide.

697 cells were cultured in medium containing 1% (vol:vol)HL1-concentrate (A) or 5% (vol:vol) of three different lots of 68°C.-treated FBS (B, C, D). Each serum preparation was contrasted before(ND) and after (D) extensive dialysis. TI-AS (+) and TI-S (−)oligodeoxynucleotides were added to replicate cultures at 200 μM fornormal oxygen-based oligodeoxynucleotides (OXY) and at 12.5 uM forphosphorothioate oligodeoxynucleotides (PT). Relative levels of DNAsynthesis (kcpm) were measured after 2 or 4 days of culture for normaland phosphorothioate oligodeoxynucleotides, respectively.

For the three different lots of FBS tested, two exhibited little changeafter dialysis in cultures containing either normal or phosphorothioateoligodeoxynucleotides. One lot of FBS, however, appeared to interfereless with the inhibitory activities of these antisenseoligodeoxynucleotides after dialysis.

EXAMPLE 11 Experiments with Stably Transfected NIH 3T3 Cells

Though the antisense oligodeoxynucleotides described herein weredesigned to block bcl-2 tRNA translation (TI-AS) and splicing (SD-AS),the molecular mechanisms of their actions are not yet known. Todetermine the effect of formation of oligodeoxynucleotide-RNA hybridswithin cells upon inhibition of cellular growth, irrespective of thenucleotide sequence, cells transformed to express human bcl-2 cDNAtranscripts were cultured with normal oligodeoxynucleotides.

200 μM of normal TI-AS and TI-S oligodeoxynucleotides were added tocultures of typical NIH 3T3 cells and to cultures of these cells thathad been stably transfected with expression constructs that produce highlevels of human bcl-2 cDNA transcripts for either the usual sense(3T3-alpha-S cells) or the antisense (3T3-alpha-AS cells) strand.

For RNA blot analyses, polyadenylated mRNA was purified from normal NIH3T3 cells and from cells stably transfected with expression constructsthat produce either sense (3T3-alpha-S) or antisense (3T3-alpha-AS)recombinant bcl-2-alpha mRNAs, according to the method of 13.Approximately 0.5 μg of mRNA was subjected to RNA blot analysis,essentially as described in (16), using either ³ _(2P)-labeledhybridization probes derived from human or murine bcl-2 sequences.

An autoradiogram resulting from a one-day exposure of a blot containingRNAs from normal 3T3 cells, 3T3-alpha-AS cells, and 3T3-alpha-S cellsshowed high relative levels of recombinant 2.4 and 1.4 kbp bcl-2transcripts produced from the bcl-2 expression constructs that weretransfected into 3T3-alpha-AS and 3T3-alpha-cells.

A 10-day exposure of a blot containing RNA from normal 3T3 cells thatwere either proliferating or quiescent at the time of harvesting RNAshowed low but detectable levels of normal 7.5 and 2.4 kbp murine bcl-2transcripts present in proliferating 3T3 cells.

TI-AS oligodeoxynucleotide specifically suppressed DNA synthesis andcellular replication in cultures of normal NIH 3T3 cells, consistentwith findings by others that fibroblasts do contain bcl-2 transcripts,albeit at low levels. The TI-AS oligodeoxynucleotide disclosed herein iscomplementary to the mouse bcl-2 sequence in 18 of its 20 bases (17),accounting for its ability to suppress the growth of murine NIH 3T3cells.

NIH 3T3 cells, 3T3-alpha-AS cells, and 3T3-alpha-S cells were culturedin medium containing 5% (vol:vol) 68° C.-treated serum and either HBSS,200 μM TI-S normal oligodeoxynucleotide, or 200 μM TI-AS normaloligodeoxynucleotide. Relative levels of DNA synthesis (kcpm) weremeasured in cultures after 3 days and reflect a 16 hour incubation with0.5 μci/well of [³H]-thymidine. Cell densities, estimated by phasemicroscopy, were consistent with the measured DNA synthesis in cultures.The percentage of inhibition of DNA synthesis in cultures containingTI-AS oligodeoxynucleotides was calculated relative to control culturescontaining HBSS.

As with normal NIH 3T3 cells, culturing 3T3-alpha-S cells (producinghuman bcl-2-alpha sense transcripts) with TI-AS and TI-Soligodeoxynucleotides demonstrated specific suppression, since the senseoligodeoxynucleotide TI-S was not inhibitory. The level of inhibition ofcellular proliferation by the antisense oligodeoxynucleotide, however,was not as great in 3T3-alpha-S cells, as might be expected, since thesecells contain more bcl-2 mRNA.

Adding TI-S oligodeoxynucleotide to cultures or 3T3-alpha-AS cells(produce antisense bcl-2 transcripts) ruled out inhibition of cellulargrowth through a nonspecific mechanism involvingoligodeoxynucleotide-RNA hybrid formation. The TI-S oligodeoxynucleotidecaused little suppression of 3T3-alpha-AS cell proliferation, whereasthe TI-AS oligodeoxynucleotide was markedly inhibitory in these cells.Similar data were obtained with TI-AS and TI-S phosphorothioateoligodeoxynucleotides.

EXAMPLE 12 Measurements of DNA Fragmentation as an Indicator of bcl-2Antisense Oligodeoxynucleotide-Mediated Programmed Cell Death in HumanLymphoma Cels

Oligonucleotides having the sequences shown in Table 2 were tested forthe ability to induce programmed cell death (DNA fragmentation) in thehuman t(14:18)-containing human lymphoma cell line RS11846. Theoligonucleotides were all phosphodiesters, and were targeted against thetranslation initiation site or the 5′-cap region of bcl-2 pre-RNAs.Control oligodeoxynucleotides included a bcl-2 sense version (TI-S) ofTI-AS (having SEQ ID NO: 7) and a scrambled version of TI-AS that hasthe same base composition, but with jumbled nucleotide order. TABLE 2SEQUENCE SEQ ID NO: CGCGTGCGAC CCTCTTG 8 TACCGCGTGC GACCCTC 9 CCTTCCTACCGCGTGCG 11 GACCCTTCCT ACCGCGT 12 GGAGACCCTT CCTACCG 13 GCGGCGGCAG CGCGG14 CGGCGGGGCG ACGGA 15 CGGGAGCGCG GCGGGC 16

RS11846 cells were adapted z crow in HL1 media with 1% FCS and their DNAmetabolically labeled by addition of ¹²⁵I-deoxyuridine to cultures orthree hours. Labeled cells were then washed thoroughly and cultured fortwo days in the presence of various oligonucleotides at 50 AM. Cellswere then recovered from 200 μL cultures by centrifugation, and lysed ina hypotonic buffer containing 10 mM EDTA and 14 Triton X100. Aftercentrifugation at 16,000×g to pellet unfragmented genomic DNA, thesupernatant fraction containing fragmented DNA was extracted withphenol/chloroform and ethanol precipitated. This DNA was then subjectedto gel electrophoresis in 1.5% agarose gel and transferred to nylonmembranes for autoradiography.

The results of two experiments are shown in FIGS. 3 and 4. The six bcl-2antisense oligonucleotides targeted in the vicinity of the ATG site oftranslation initiation in bcl-2 mRNAs were tested. “C-Oligo-2” refers toan oligonucleotide with 4 purposeful mismatches. “U” indicates untreatedcontrol cells. FIG. 4 shows the results for the oligonucleotides shownin FIG. 3. “Sc2C” refers ta a 20 mer with the same base composition asTI-AS, but with scrambled sequence. FIG. 4(b) shows the results forthree oligonucleotides targeted against the 5′-cap of bcl-2 mRNAs. Thenumbers refer to the distance of these oligomer from he ATG-translationinitiation=site.

The presence of a ladder of DNA fragments (unit size of approximately200 bp) is indicative of programmed cell death. At 50 μM, TI-AS causedlittle DNA fragmentation, whereas the oligonucleotides having SEQ ID NO:9 and SEQ ID NO: 10, and one of the 5′-cap oligonucleotides (SEQ ID NO:14) led to pronounced DNA Fragmentation.

EXAMPLE 13 Concentration-Dependence of Inhibition by AntisensePhosphodiester Oligodeoxynucleotides in Serum-Free Cultures

697 pre-3 cell leukemia cells were cultured in medium with either 1%(vol:vol) HL-1 concentrate (serum-free conditions [o]3% (vol:vol) 68°C.-treated serum (FBS2) [______], see FIG. 5. Shown are cellulardensities measured after 2 days in cultures containing variousconcentrations of phosphodiester TI-AS oligodeoxynucleotide. Data areshown as percentages relative to control cultures treated with a senseoligonucleotide, and reflect the mean±standard deviation for duplicatesamples.

EXAMPLE 14 Immunofluorescence Analysis of bcl-2 Protein Levels inOligodeoxynucleotide-Treated 697 Cells

For studies with oligodeoxynucleotides, 0.25×10⁴ (for phosphorothioate)or 0.5×10⁵ (for normal oligodeoxynucleotides), 697 cells were culturedin 1 ml of HL-1 serum-free medium. in 24 well culture dishes (Linbro.Flow Lab, Inc.). After 2 days (for normal) or 4 days (forphosphorothioates), cells were recovered from cultures, washed once inPBS, pH 7.4 (Gibco)—0.1% bovine serum albumin—0.1% sodium azide], andfixed for 5-10 minutes on ice in 1% paraformaldehyde/PBS solution. Thecells were then washed once in PBS and incubated in 1 ml of absolutemethanol at 20° C. for 10 minutes. After washing once in PBS-A, cellswere then resuspended in PBS containing 0.05% Triton-X100 for 3 minuteson ice, washed in PBS-A and preblocked for 30 minutes at 4° C. in PBSwith 10% (v/v) heat-inactivated goat serum.

For addition of he first antibody, preblocked cells were resuspended in100 μl of PBS-G (PBS-1% goat serum-0.1% sodium azide) prior toaliquoting 50 μl into separate tubes that contained 1 μl of either BCL2antibody (Halder et al., Nature (London), 342: 195-197 (1989)) oraffinity-purified normal rabbit control IcG (Cappel 6012-0080) andincubated for 1 hour on ice. The BCL2 antibody used war these studieswas prepared in rabbits using a synthetic peptide corresponding to aminoacids (98-114) of the BCL2 protein and was affinity—purified byprotein-A-Sepharose chromatography and used at approximately 1 mg/ml.Cells were then washed in PBS-A and incubated in 0.5-1.0 ml PBS-A for15-20 minutes on ice to allow diffusion of nonspecific cell-associatedantibody prior to resuspending cells in 100 μl of PBS-G containing 5 μgof biotinylated scat anti-rabbit IgG (BAIOOO; Vector Labs) for 30minutes. After washing once and incubating for 15 minutes in PBS-A,cells were finally resuspended in 100 μl of PBS-A containing 2 μg ofFITC-conjugated avidin (Vector Labs A 2011) for 20 minutes and washedthree times in PBS-A prior to analysis with an Ortho cytofluorograph50-H connected to an Ortho 2150 data-handling system. The specificity ofmethod for detecting BCL2 protein was confirmed by immunofluorescencemicroscopy (showing cytosolic stain peptide competition, and studies ofcell lines that expressed various levels of BCL2 mRNA and proteinsthrough gene transfer manipulations.

For measurements of surface HLA-DR antigen expression, an indirectimmunofluorescence assay method was used (Reed et al., J. Immunol134:1631-1639 (1985)) involving incubation of viable cells with a murineanti-HLA-DR monoclonal antibody ((IgG2a) (Becton-Dickinson 7360) or anegative control antibody, R3-367 (IgG2a), followed by FITC-conjugatedscat anti-mouse IgG (Cappel 1711-0081). Cells were fixed in 1%paraformaldehyde/PBS prior to FACS analysis.

697 cells were cultured for days 2 days (PO) or 4 days (PS) with variousoligonucleotides. In FIG. 6, the black columns show the results with asense oligonucleotide, and the hatched columns with an antisenseoligonucleotide TI-AS. Cells were labeled with anti-bcl-2 antiserum andanalyzed by FACS. Data are expressed as percentages relative to the meanfluorescence obtained with untreated 697 cells.

FIG. 7 shows typical FACS results obtained for 697 cells before andafter treatment with 100 μM PO bcl-2 antisense oligonucleotides. A:untreated 697 cells labeled with either anti-bcl-2 antiserum (hatchedarea) or normal rabbit serum control (white area); B: untreated 697cells labeled with either anti-HLA-DR antibody (hatched area) or anegative control antibody (white area); C: 697 cells cultured for 2 dayswith either normal bcl-2 TI-AS (white area) or TI-AS (hatched area)oligodeoxynucleotides and labeled with anti-bcl-2 antibody; D: 697 cellscultured with TI-AS and TI-S oligodeoxynucleotides (as in C), butlabeled with anti-HLA-DR antibody.

As shown in FIGS. 6(a) and (b), PO and PS bcl-2 antisenseoligonucleotides produced specific concentration-dependent reductions inthe levels of bcl-2 proteins, without altering the levels of expressionof HLA-DR (FIG.—7) and other control antigens. At 150 μM, for example,PO antisense oligodeoxynucleotide caused an approximately 75-95%reduction in bcl-2 fluorescence, whereas the control senseoligodeoxynucleotide diminished bcl-2 protein levels by only 10-20%(FIG. 6(a)). similarly, cultured 697 cells for 4 days with the PSantisense oligodeoxynucleotide ar 25 μM resulted in approximately 70%reduction in bcl-2 fluorescence. In comparison, the sense PSoligodeoxynucleotide TI-AS inhibited bcl-2 protein levels by onlyapproximately 15%, as measured by this assay (FIG. 6(b)).

Significance

In phosphorothioate oligodeoxynucleotides, one of the non-bridgingoxygen atoms in each internucleotide phosphate linkage is resolaced by asulfur atom. This modification renders phosphorothioateoligodeoxynucleotides extremely resistant to cleavage by nucleases,Stein et al., Nucl. Acids Res., 16:3209-3221 (1988). Despite thesubstitution of a sulfur atom for an oxygen, phosphorothioateoligodeoxynucleotides retain good solubility in aqueous solutions;hybridize well, though with some decrease in the melting temperature ofRNA-oligodeoxynucleotides duplexes; and are synthesized conveniently bythe widely employed method of automated oligodeoxynucleotides synthesiswith phosphoroamidites.

Antisense bcl-2 phosphorothioate oligodeoxynucleotides have been foundto be more potent inhibitors of leukemic cell grown than their normaloxygen-based counterparts. When tested under serum-free conditions,these oligodeoxynucleotides reduced cellular proliferation by half atconcentrations of approximately 15-23 μM, whereas the normaloligodeoxynucleotide achieved 50% inhibition at 125-250 μM. This findingmay be explained by the reduced sensitivity phosphorothioateoligodeoxynucleotides to cellular nucleases, or may be attributable toother mechanisms. For example, mRNAs hybridized with phosphorothioateoligodeoxynucleotides may experience enhanced degradation through amechanism involving an RNAse H-like activity.

Despite their increased inhibitory activity, phosphorathioate antisenseoligodeoxynucleotides retained sequence-specificity. A: theconcentrations tested (less than 25 μM), sense versions of theseoligodeoxynucleotides had little effect on leukemic cell growth. Bothnormal and phosphorothioate antisense oligodeoxynucleotides appearedinitially suppress the proliferation of leukemic cells throughnon-cytotoxic mechanisms. During the first few days of culture, cellularreplication was inhibited without a concomitant rise in cell death.Later in these cultures days 4-5 for normal oligodeoxynucleotides, days6-8 for phosphorothioates), however, cellular viabilities declined.

Comparing the kinetics of inhibition by normal and phosphorothioateoligodeoxynucleotides revealed that the latter compounds have a sloweronset of action. Maximal inhibition of leukemic cell proliferation bynormal antisense oligodeoxynucleotides occurred two days afterinitiation of cultures, whereas phosphorothioate oligodeoxynucleotidesrequired 4 to 7 days to achieve maximal inhibition.

The usefulness of anticode oligomers in inhibiting humanlymphoma/leukemia cells and other types of cancer cells that express thebcl-2 gene has been shown by the examples herein. Anti-senseoligodeoxynucleotides complementary to at least an effective portion ofthe mRNA of the human bcl-2 gene has been found to inhibit growth ofRS11846 human follicular lymphoma cells t (14;18) chromosomaltranslation and high bcl-2 expression), 697 human pre B cell leukemiacells (high bcl-2 expression), JURKAT human acute lymphocytic leukemiacells (medium bcl-2 expression) normal human lymphocytes (medium bcl-2expression), and murine fibroblasts (low bcl-2 expression). Althoughbcl-2 antisense reagents can suppress the growth of many types of cells,the t(14:18) lymphoma and leukemia cells seem to be the sensitive,allowing for specific inhibition of malignant cells.

As demonstrated in the following Examples, a variety of DNA analogs canbe employed in the instant invention. For example, phosphorothioates,methylphosphonates, and mixed oligomers containing combinations ofphosphodiesters and phosphorothioate or methylphosphonate nucleosides.It should be understood that RNA analogs can also be employed in theinvention.

EXAMPLE 15 Methylphosphonate (MP)/Phosphodiester (PO) bcl-2 AntisenseOligomers Induce Death of DoHH2 Lymphoma Cells

The purpose of this study was to determine the efficacy of variousanalogs of the anticode oligomers for inhibiting lymphoma cell survival.

DoHH2 is a human lymphoma cell line containing a t(14:18)-translocationthat activates the bcl-2 gene. DoHH2 cells were cultured for 3 dayswithout oligomers or in the presence of various concentrations ofantisense (As) and scrambled (Sc) methylphophonate (MP)/Phosphodiester(PO) oligomers for 3 days. Cell viablity was assessed by trypan blue dyeexclusions, and the data expressed as a percentage relative to DoHH2cells cultured without oligomers. The MP/PO oligomers was an 18-mertargeted against the first 6 codons of the bcl-2 open reading frame inwhich 5 internal linkages were phosphodiester and the flankingnucleosides were methylphophonates.

The results indicate that these anticode oligomer analogs are potent andspecific inhibitors c. lymphoma cell survival.

EXAMPLE 16 Methylphosphonate (MP)/Phosphodiester (PO) Chimeric OligomersInhibit Growth of MCF-7 Human Breast Cancer Cells

The purpose of this study was to determine the efficacy of the claimedanticode oligomer analogs to inhibit the survival of solid tumor cellswhich highly express bcl-2.

MCF-7 is a human breast adenocarcinoma cell line that containsrelatively high levels of bcl-2 protein. The cells were cultured at4,000 cells per well in 96-well microtiter plates in the presence orabsence of MP/PO oligomers. Relative cell numbers per well were thenestimated by MTT assay, based on a standard curve prepared using freshlyplated, untreated MCF-7 cells. The antisense (As) and scrambled (Sc)MP/PO oligomers wer the same as those described in Example 16. Datarepresent the mean+/−standard deviation for determinations.

The results demonstrate sequence specific inhibition of growth of solidtumor cells by the the claimed anticode oligomer analogs.

EXAMPLE 17 Optimimization of Anticode bcl-2 Oligomer Sequences

The purpose of this study was to determine optimum target sites orsequence portions on mRNA for inhibiting cell survival by contacting thecells with various claimed anticode molecules whose sequences werecomputer generated.

DoHH2 lymphoma cells were treated with various concentrations ofoligomers targeted to different sites on the bcl-2 mRNA. The ATGoligomer targets the translation initiation site, and is complementaryto the first 6 codons of the open reading frame. The Dscore 23 andDscore 72 oligomers target sites in the 5′ untranslated region of themRNA. Sc oligomers represent negative controls having the same lengthand base composition but in scrambled order. All oligomers were preparedas phosphodiester (PO)/phosphorothioate (PS) chimeras, where only thelast (3′) two internucleoside linkages were phosphorothioates. oligomerswere added directly to cultures and relative numbers of viable cellswere estimated by MTT assay 3 days later. Data represent mean+/−standarddeviation.

The results indicate that the Dscore 23 oligomer, targeted to the 5′untranslated region, has, compared to the other anticode oligomerstested in this Example, superior activity for inhibiting cell survival.

EXAMPLE 18 Reveral of Chemoresistance of Tumor Cells byAntisense-Mediated Reduction of bcl-2 Gene Expression

The following work was undertaken to determine if anticode oligomersdirected against the expression of the bcl-2 gene would reversechemoresistance, that is to say, increase the sensitivity to cancerchemotherapeutic agents in cancer tumor cells expressing the bcl-2 gene.

High levels of bcl-2 protein appeared to increase the relativeresistance of lymphoid cells to killing induced by a wide variety ofcancer chemotherapeutic agents including, but not limited to, Ara-C,MTX, vincristine, taxol, cisplatin, adriamycin, etoposide, mitozantron,2-chlorodeoxyadenosine, dexamethasone (DEX), and alkylating agents.(Miyashita, T. and Reed, J. C., Cancer Res. 52:5407, Oct. 1, 1992).While these drugs have diverse biochemical mechanisms of action, it isbelieved that all have in common he ability to ultimately trigger cancercell death by activating endogenous cellular pathways leading toapoptosis (Eastman, A. Cancer Cells 2:275 (1990)). It is understood thatthe claimed anticode molecules and analogs thereof as used herein areeffective for their intended purposes Of enhancing sensitivity to cancerchemotherapeutic drugs including, but not limited to, antimetabolites,alkylating agents, plant alkaloids, and antibiotics.

Antimetabolites include, but are not limited to, methotrexate,5-fluoruracil, 6-mercaptopurine, cytosine arabinoside, hydroxyurea,20chlorodeoxy adenosine.

Alkylating agents include, but are not limited to, cyclophospham; ide,melphalan, busulfan, cisplatin, paraplatin, chlorambucil, and nitrogenmustards.

Plant alkaloids include, but are not limited to, vincristine,vinblastine, VP-16.

Antibiotics include, but are not limited to, doxorubicin (adriamycin),daunorubicin, mitomycin c, bleomycin.

Other cancer chemotherapeutic agents include DTIC (decarbazine), mAMSA,hexanethyl melamine, mitroxantrone, taxol, etoposide, dexamethasone.

In the present work both nuclease resistance phosphorothioates (PS) andphosphediesters in which only the 3′-most internucleoside bond was athioate linkage (PO/PS) were employed. The PO/PS oligomers areresistance to 3′ exonucleases (the principal nuclease activity of serum)and generally form more stable heteroduplexes with target RNAs.

Cationic lipids were used to improve the uptake and subsequent releaseor oligomers into effective Intracellular compartments, and areexemplary pharmaceutical carriers for the claimed anticode oligomers.

The methods for preparing and purifiying the antisense (AS) andscrambled (SC) 18′mer oligonucleotides used for the present work aredescribed above in General Methods and in Kitada et al. (Antisense R &D, 3:157 (1993)). Phosphodiester oligonucleotides were synthesized in a10-15 micromole scale using phosphoroamidate chemistry with oxidation byiodine, and then purified using a C₁₈-reverse phase column. In mostcases, oligomers were additionally ethanol-precipitated five times toeliminate any nonspecific cytotoxic activity, and then dried andresuspended in sterile HL-1 medium (Ventrex Labs, Inc; Burlingame,Calif.) at 1-10 mM. The pH of this solution was adjusted using 1-10 MNaOH until the phenol red indicator dye in the media returned to itsoriginal color.

The principal oligomers used were 18-mers, having either the sequence:

-   -   I. TCTCCCAGCGTGCGCCAT (SEQ ID NO. 17), which is antisense to the        first six codons of the human bcl-2 open reading frame (SEQ ID        NO. 19); or    -   II. TGCACTCACGCTCGGCCT (SEQ ID NO. 18), which is a scrambled        version used as a control.

Standard transfection methods were used to produce tumor cellsexpressing either the bcl-2 gene or an antisense oligodeoxynucleotidewhich bound to bcl-2 mRNA. It is understood that the vector could alsoencode an antisense oligodeoxynucleotide which binds to bcl-2 pre-mRNA.The particular nucleotide sequence encoding the antisenseoligonucleotides of the invention is not critical, except that thesequences are preferably chosen such that they express antisenseoligodeoxynucleotides sufficient to reduce bcl-2 gene expression intumor cells and increase the sensitivity of the tumor cells to cancerchemotherapeutic agents or sufficient to kill tumor cells when they aretreated with cancer chemotherapeutic agents. It is only necessary thatthe antisense oligodeoxynucleotide encoded in vector is expressed underconditions sufficient to reduce bcl-2 gene expression in tumor cells.The methods used for preparing vectors, and, in particular, expressionplasmids, for transferring genes into mammalian cells relies on routinetechniques in the field of molecular biology. A basic text disclosingthe general methods of preparing expression plasmids used in thisinvention is Molecular Cloning, A Laboratory Manual, 2nd Editon, eds.Sambrook et al., Cold Spring Harbor Laboratory Press, (1989),particularly Chapter 16 on Expression of Cloned Genes in CulturedMammalian Cells. Examples 15C-D below set forth particular methods forpreparing the expression plasmids used in the present invention. Theparticular vector used to transfer the antisense oligonucleotides of thepresent invention is not critical, and such vectors may include vectorsderived from lambda and related phages or from filamentous phages. It isonly necessary that the transfered nucleotide sequence encoding theantisense oligonucleotides of the present invention be expressed in thetransfected tumor cell under conditions sufficient to reduce bcl-2 geneexpression in the tumor cell. The present invention includes expressionof the antisense oligonucleotide either from an extrachromosomalposition (e.g. from an expression plasmid) or from a position integratedinto the host genome itself, as mediated by other vectors, such asrecombinant retroviral vectors (Reed et al. bcl-2 mediatedtumorigenicity in a T-cell lymphoid cell line: synergy with C-MYC andinhibition by bcl-2 antisense. PNAS USA 87:3660 (1990)).

A. Treatment of Lymphoma Cells with 18-mer Synthetic bcl-2 Antisenseoligodeoxynucleotides.

Lymphoma cell line SU-DHL-4, obtained from a use of diffuse,histiocytic, non-Hodgins lymphoma (Epstein et al. Two new monoclonalantibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embeddedtissues with follicular center and mantle zone human B lymphocytes andderived tumors. J. Immunol. 133:1028 (1984)) and containing a t(14;18)translocation was treated with 18-mer synthetic bcl-2-ASoligodeoxynucleotides targeted for binding with the first six codons ofthe bcl-2 mRNA. As a control, SU-DHL-4 cells were treated with variouscontrol oligomers, including 18-mers having the same nucleosidecomposition as the AS oligomer, but in which the bases were in scrambledorder (SC).

Aliquots of 1.5 ml of HL-1 serum-free medium (Ventrex Labs, Inc.)supplemented with 1 mM L-glutamine, 50 Units/ml penicillin, and 100ug/ml streptomycin and either 5 ug of purified oligonucleotides or 30 ugof Lipofectin³ [1:1 w/w mixture ofN-(1-2,3-dioleyloxy)propyl)-n,n,n-trimethylammonium chloride (DOTMA) anddioleoylphophotidylethanolamine (DOPE)] were combined and added to0.75×10⁶ SU-DHL-4 cells in 3 mls of HL-1 medium. Cells were then eithercultured at 37° C. in a humidified atmosphere of 5% CO₂/95% air in 24well plates (2 mls/well) for immunoblot and RT-PCR assays, or in 96-wellflat-bottom microtiter plates (0.1 ml/well) for MTT assays. For cells inmicrotiter cultures, typically 0.1 ml of additional HL-1 media with orwithout various chemotherapeutic drugs was added after 1 day, and thecells were cultured for an additional 2 days before performing MTTassays.

Cells were washed once in PBS, lysed in a buffer containing 1% TritonX100, and samples normalized for protein content (25 ug) prior tosize-fractionation of proteins by SDS-PAGE (12% gels) and transfer tonitrocellulose filters for immunoblot assays as described in Reed et al.Cancer Res. 51:6529 (1991). Preliminary experiments determined thataliquots of lysates containing 25 ug of total protein produced resultsin the linear range of the assay. Blots were first incubated with 0.1%(v.v) of a rabbit antiserum directed against a synthetic peptidecorresponding to amino-acids (aa) 41-54 of the human Bcl-2 pre-ein, asshown in SEQ ID NO. 21 (id) followed by 2.8 ug/ml biotinylated goatanti-rabbit IgG (Vector Labs, Inc.). Bands corresponding to p26-Bcl-2were then visualized by color development using a Horseradish Peroxidase(HRP)-avidin-biotin complex reagent (Vector Labs, Inc) and3,3′-diaminobenzidine (DAB). Stained blots were then incubated with asecond anti-Bcl-2 antibody directed against aa 61-76 of the Bcl-2protein (SEQ ID NO. 21) followed by 0.25 uCi/ml ¹²⁵l-protein A. Bcl-2bands were excised from the blots and subjected to gamma-counting.

Despite the mitochondrial location of Bcl-2 protein, difference in therate of MTT dye reduction by mitochondrial enzymes was noted in cellstwat were identical except for their levels of p26-Bcl-2. Thesecomparisons were made using pairs of exponentially growing lymphoid celllines that differed only in that one line had been stably infected witha recombinant bcl-2 retrovirus and the other with the parentalretroviral vector lacking a bcl-2 cDNA insert (Miyashita et al. CancerRes. 52:5407 (1992); Blood 81:171 (1993)).

Anticode specific reductions in the relative Levels of bcl-2 mRNA weredetected within 1 day by a semi-quantitative reverse transcriptasepolylmerase chain reaction (RT-?C.) assay. See FIG. 3A.

SU-DHL-4 cells were cultured with 0.83 ug/ml of oligomers complexed with5 ug of cationic lipids (Lipofectin; BRL/Gibco, Inc.) per ml ofserum-free media y(13,19). In FIG. 5A, total RNA was isolated from cellsafter 1 day and relative levels of bcl-2 and glyceraldehyde 3-phosphatedehydrogenase (GAPDH) mRNAs were assessed by RT-PCR assay as describedin Kitada et al. Antisense R & D 3:157 (1993)).

In FIG. 8B, SU-DHL-4 cells were cultured with pairs of either PS(squares) or P0/PS (circles) As- and Sc-Oligomers for 3 days. Relativelevels of Bcl-2 protein were then measured using a quantitativeimmunoblot assay, as described above, and the data expressed as apercentage relative to cells treated with control Sc-oligomers. Theinset shows immunoblot results for p26-Bcl-2 and a p75 cross-reactive(CR) band in a case where As-PO/PS oligomer produced a 41% relativedecrease in Bcl-2 protein levels. In FIG. 8C, 10⁻⁴M Ara-C, MTX, or DEXwas added 1 day after addition of PS (squares) or PO/PS (circles)oligomers to cultures of SU-DHL-4 cells, and MTT assays were performedon day 3. Data are presented as a % control relative to cells culturedwith drugs in the absence of any oligomers, and represent the results of9 of 10 consecutive experiments [in one experiment, the MTT assayfailed]. Similar results were obtained when dye exclusion assays wereused to assess cell survival rather than MTT assay [not shown].

Mean values for the data are indicated by horizontal lines. Statisticalanalysis o: the data was by paired t-test (As versus Sc). Concentrationsof As- and Sc-oligomers (≈150 nM) were adjusted ta maximize As effectswhile maintaining sequence specificity.

Variations in the amounts of starting RNA were controlled for by RT-PCRanalysis using primers specific for GAPDH RNA.

The long half-life of the bcl-2 protein (approximately 14 hours) mayaccount for the AS-mediated reductions in bcl-2 proteins not being asdramatic as for reductions in bcl-2 mRNA, taking longer to achieve(about 3 days), and appearing more variable.

FIG. 8B shows the composite results for 10 experiments where relativelevels of bcl-2 protein were compared in SU-DHL-4 cells treated with ASor SC oligomers. AS-mediated reductions in bcl-2 protein levels rangedfrom as much as 66% to as little as 10%, with an average relativereduction of about 30%, compared to SU-DHL-4 cells that were treated inthe identical manner with control oligomers. Levels of a variety ofcontrol mitochonrial proteins such as F₁-beta-ATPase and cytochrome C,which like bcl-2 are encoded by nuclear genes, were not adverselyaffected by AS-oligomers (not shown), indicating that the AS-mediatedreductions in bcl-2 protein levels were specific. The insert in FIG. 8B,for example, shows a comparison of p26-Bcl-2 with a 78-kDa protein thatcross reacts with one of the rabbit antisera employed for immunoblotassays, demonstrating a decrease in the levels of p26-bcl-2 but not p78in the AS-treated cells relative to cells that received controlSC-oligomers.

B. Effect of Treatment of SU-DHL-4 Cells with bcl-2 AS Oligomers on CellSensitivity to Cancer Chemotherapeutic Agents

This study was performed to determine whether treatment of SU-DHL-4cells with bcl-2 AS-oligomers could increase their relative sensitivityto killing by the cancer chemotherapeutic agents Ara-C, MTX, and DEX,which are anticancer drugs.

Previous control studies demonstrated that bcl-2 AS oligomers had littleor no effect on SU-DHL-4 cell growth and survival at least during thefirst three days of culture (Kitada et al. Antisense R & D 3:157(1993)). AS-mediated reductions in bcl-2 protein levels in theselymphoma cells as well as in other cells do no typically accelerate therate of cell death in cultures unless the cells are deprived of serumgrowth factors (Peed et al. Proc. Natl. Acad. Sci. USA 87:3660 (1990)).

In the present work, preliminary studies demonstrated that more than 90%of SU-DHL-4 cells survived treatment for 4 days with high dose (10 ⁻⁴)Ara-C, MTX or DEX, presumably because of their high levels of bcl-2protein (Not shown). At these concentrations, however, all drugs inducedessentially complete inhibition of SU-DHL-4 cell proliferation,consistent with bcl-2 converts drugs from cytotoxic to cytostatic.Comparisons or AS and SC oligomers demonstrated that bcl-2 AS treatmentmarkedly enhanced the sensitivity of these lymphoma cells to MTX andAra-C, and to a lesser extent to DEX (FIG. 3C).

Despite some variability in results, on average, the addition of bcl-2AS oligomers to cultures of SU-DHL-4 cells treated with MTX or Ara-Cresulted in 79-84% greater inhibition (reduction in viable cell numbers)than use of either drug alone (?<0.002 for AS versus SC) in the absenceof introducing the bcl-2 AS oligomers of the invention. Statisticallysignificant results were obtained for DEX-treated SU-DHL-4 cells(P=0.01). The 20-304 reduction in viable cell numbers observed forcontrol oligomer-treated cells could reflect a degree of sequencenon-specificity but was probably related to the use of cationic lipidsto facilitate oligomer delivery into cells.

C. Effect of Transfecting Cells with Expression Plasmids Encoding Humanbcl-2 Protein on Sensitivity to Chemotherapeutic Agents.

To further confirm the sequence specificity of bcl-2 AS oligomers forenhancing sensitivity to chemotherapeutic anticancer drugs, a study wasconducted using an Interleukin-3 (IL-3)-dependent murine hemopoieticcell line 32D.C 13 that had been stably transfected with expressionplasmid encoding either the human bcl-2 protein or a viral homolog ofbcl-2, BHRF-1, which has only 22% homology with bcl-2. 32D.C13 cellswere obtained from Dr. Giovanni Rovera of the Wistar Institute,Philadelphia, Pa.

Treatment of 32D cells with oligomer/cationic lipid complexes was asdescribed above except that 50 Units/ml of murine recombinant IL-3(rlL-3) was included in the HL-1 media, the initial cell density was 10⁵per ml, and replication-defective adenovirus dl3l2 (MOI=200) was added30 minutes after exposure of cells to oligomers to facilitate exit ofDNA from endosomes [Yoshimura K, et al. J Biol. Chem. 268, 2300,(1993)].

32D cells that had been stable transfected with expression plasmidsencoding either human p26-Bcl-2 or EBV p19-BHRF-1 (Takayama, S. et al.submitted) were cultured in medium (10⁵/ml) containing IL-3 and PO/PSoligomers for 3 days to achieve reductions in human Bcl-2 proteinlevels. The cells were then retreated with oligomers alone (C) or incombination with various concentrations of MTX and the relative numberof viable cells assessed by MTT assay 2 days later. Data representmean+/−standard deviation for triplicate determinations and areexpressed as a % relative to cells that received no MTX. Statisticalanalysis of data for 10⁻⁶ to 10⁻⁴ M MTX was by a 2-way Analysis ofVariables method (Finney, D. J. In Statistical Methods in BiologicalAssays, p. 72, 1978 (3rd edition, Charles Griffin & Co., London).Comparable results were obtained with dye exclusion assays [not shown].

RNAs derived from the human bcl-2 construct in 32D-BCL-2 cells were atarget for bcl-2 AS oligomers, whereas RNAs from the BHRF-1 expressionplasmid are not. Thus the chemosensitivity to cytoxic drugs of 32D.C13cells expressing BHRF-1 should have been unaffected by the AS treatment.

Preliminary experiments demonstrated that upon withdrawal of IL-3 from32D.C13 cells, levels of endogenous mouse bcl-2 protein declined and thecells underwent apoptosis. bcl-2 and BHRF-1 comparably supported thesurvival of 32D.C13 cells in the absence of IL-3, and the proliferativerates of 32D.C13 cells containing high levels of these proteins weresimilar in the presence of IL-3, thus excluding these variables asexplanations for any differences in chemosensitivity.

FIG. 9 compares the sensitivity of 32D-BCL-2 and 32D-BHRF-1 cells tovarious concentrations of MTX. Treatment with bcl-2 AS-oligomersresulted in sequence-specific increases in the sensitivity of 32D-BCL-2cells to inhibition by MTX at concentrations of 10⁻⁶ to 10⁻⁴ M (P≦0.001for AS versus SC). In contrast, treatment with bcl-2 AS oligomersproduced no significant difference in the sensitivity of 32D-BHRF-1cells to MTX, relative to control SC-oligomers (FIG. 9). These dataindicate that the effects of bcl-2 AS oligomers on chemosensitivity tocytoxic agents drugs are sequence specific. Furthermore, several othercontrol oligomer, including bcl-2 sense, other scrambled sequences withthe same nucleoside composition as AS, and oligomers with totallyunrelated sequences all had comparatively little effect on thechemosensitivity of the cells (Not shown).

The findings above demonstrated that bcl-2 AS oligomers producedsequence specific reductions in bcl-2 mRNA and bcl-2 protein levels andthat these events were associated with increased sensitivity tochemotherapeutic agents such as anticancer drugs. The portion of tumorcells killed by the chemotherapeutic agents was greater than the portionkilled by the same amount of chemotherapeutic agents in the absence ofintroducing the bcl-2 AS oligomers of the invention.

D. Effect of Transfecting Cells with Expression Plasmids Encoding Humanbcl-2 Protein on Sensitivity of Lymphoma Cells to ChemotherapeuticAgents.

A different strategy Was employed to determine if AS-mediated reductionsin bcl-2 gene expression could be achieved with an inducible bcl-2 ASexpression plasmid that used a heavy metal response humanmetallothionein-IIA promoter in another translocationt(14;18)-containing lymphoma line, RS11846. RS-1846 Was obtained fromDr. Carlo Croce (Wistar Institute, Philadelphia, Pa. (Tsujimoto andCroce, Proc. Natil. Acad. Sci. USA 53:5214 (1986)).

To prepare the expression plamid, a 0.91 kbp blc-2 cDNA (ibid)) wassubcloned in either antisense (AS) or sense (S) orientation into aHindIII site downstream of a human metalothionein-IIA promoter in theplasmid pMEP-4 (Invitrogen, Inc.), which contains a hygromycinphosphotransferase from and the ESNA-1 gene and origin of DNAreplication from Epstein Varr Virus for high copy episomal maintenance.

RS11846 cells (5×10°) in Dulbecco's phosphate buffered saline containing30 ug of plasmid DNA were electroporated (1500 uF, 270 V/cm) using aCellject Electroporation System from EquiBio, Inc. Cells were returnedto their usual culture media RPMI-L 1640 supplemented with 10% fetalbovine serum, 1 mM L-glutamine, 50 Units/ml penicillin, and 100 ug/mlstreptomycin) at 2×10⁵ cells per ml and cultured for 2 days beforeseeding cells at 2×10⁵ per ml in media containing 200 ug/ml hygromycin.After 3 weeks of culture, the resulting bulk cell lines were passaged insuccessively higher concentrations of hygromycin in 200 ug/ml incrementsuntil the concentration reached 1 mg/ml (about 4 weeks).

Hygromycin-resistant RS11846 cell were cultured in RPMI/10% serum mediacontaining 0.5 uM CdCl₂ and 3 days later immunoblot assays wereperformed using 25 ug protein/lane essentially as described in Tanaka S,et al. J. Biol. Chem. 268, 10920 (1993) and in Reed et al. Cancer Res.51:6529 (1991)).

As summarized in FIG. 10, control (“C”) and bcl-2-As (“As”) plasmidswere introduced into RS11846 cells and expression was induced witheither 0.5 uM CdCl₂ or 50 uM ZnCl₂ for various times. As an additionalcontrol, RS11846 cells containing inducible plasmids with the bcl-2 cDNAin sense (“S”) orientation were also analyzed. RS11846 cells wereinduced for 3 days and relative levels of Bcl-2 and F₁-βR-ATPaseproteins were assessed by immunoblot assay of Tanaka et al. J. Biol.Chem. 268:10920 (1993). In FIG. 10A, RS11846 cells were cultured at 10⁵cells/ml in medium containing 0.5 uM CdCl₂ and 1 day later 10⁻⁷ M Ara-Cor an equivalent volume of diluent control was added. Relative numbersof viable cells were estimated from MTT assays at various times and themean+/−S.D. calculated for triplicate samples. In FIG. 10B, RS11846cells were cultured as in FIG. 10A, except that various concentrationsof Ara-C, MTX, or DE were added. Data represent mean+/−S.D. fortriplicate samples. Statistical calculations are by 2-way Analysis ofVariables. DEX served as a negative control here since RS11846 cellshave lost glucocorticoid receptors.

Preliminary experiments demonstrated that RS11846 cells tolerated theaddition of up to 0.5 microM CdCl₂ or to microM ZnCl₂ to cultures forone week, experiencing a slight decrease in growth rate but essentiallyno decline in percentage cell viability (Not shown).

In the absence of heavy metal induction, the relative levels of bcl-2protein in RS11846 cells containing the control or bcl-2 AS plasmid werecomparable, as determined by immunoblot assays (Not shown). When 0.5 umCdCl₂ or uM ZnCl₂ was added, reductions in bcl-2 protein became evidentin the AS-expressing cells at 2 days and maximal inhibition of 30-40%was obtained at three to four days, relative to control RS 11846 cells.

FIG. 10A shows an example of immunoblot data derived from RS11846 cellsafter three days of exposure to 0.5 mM CdCl₂, demonstrating reducedlevels of bcl-2 protein in the AS-plasmid containing cells compared toRS11846 cells that harbored the control plasmid. The relative levels ora control mitochrondrial protein F₁-beta-ATPase were comparable in allcell lines, consistent with sequence-specific alterations in bcl-2protein levels.

When RS 11846 cells containing either the control or bcl-2-As plasmidswere cultured or various times in 0.5 uM CdCl₂ or 50 uM ZnCl ₂, nosignificant difference in the growth rates of these two cell lines wasobserved (FIG. 85). Thus, As-mediated reductions in Bcl-2 protein levelsby themselves did not impair RS11846 cell proliferation or survival.

Inclusion of low-dose Ara-C (10⁻⁷M) in the cultures of control RS11846cells resulted in only a slight decline in the net numbers of viablecells, presumably because of the high levels of Bcl-2 protein found inthese t(14;18)-containing lymphoma cells. In contrast, addition of 10⁻⁷MAra-C to cultures of bcl-2-AS expressing RS11846 cells was markedlyinhibitory (FIG. 8B). Ara-C, however, had no effect on bcl-2AS-expressing RS11846 cells in the absence of heavy metal induction ofthe MT promoter, when directly compared with RS11846 cells containingthe control plasmid under the same conditions [not shown]. FIG. 8C showsthat the enhanced sensitivity to Ara-C observed for bcl-2-AS-expressingRS11846 cells occurred over a wide range of drug concentrations(P<0.001). Heavy-metal induction of the bcl-2-AS expression plasmid alsosignificantly increased the relative sensitivity of RS11846 lymphomacells to MTX (P<0.001), but not to DEX. Glucocorticoid receptor bindingassays demonstrated that RS11846 cells have lost receptors for thesesteroid hormones [not shown], thus providing a specificity controlshowing that AS-mediated reductions in bcl-2 protein levels are bythemselves insufficient to impair the growth or survival of theselymphoma cells.

Using a plurality of anticode approaches, the present inventiondemonstrated that average reductions of 30-40% in the relative levels ofbcl-2 protein markedly enhanced the sensitivity of lymphoma cells, inparticular, t(14;18)-containing lymphoma cell lines to chemotherapeuticagents such as conventional anticancer drugs. These examplesdemonstrated that introducing the claimed anticode oligomers into tumorcells achieves a reduction of bcl-2 expression and increases thechemosensitivity of neoplastic cells to chemotherapeutic agents oranticancer drugs.

Accordingly, the present invention achieved a method of killing tumorcells by introducing to tumor cells anticode oligomers which reducebcl-2 gene expression or impair Bcl-2 protein function before contactingthe cells with chemotherapeutic agents including anticancer drugs. Theconventional anticancer drugs reduced the numbers of viable malignantcells, and the portion of tumor cells killed was greater than theportion which would have been killed by the same amount of drug in theabsence of introducing the anticode oligomers into the cells.

Having thus disclosed exemplary embodiments of the present invention, itshould be noted by those skilled in the art that this disclosure isexemplary only and that various other alternatives, adaptations, andmodifications may he made within the scope of the present invention.Accordingly, the present invention is not limited to the specificemobodiments as illustrated herein, but is only limited by the followingclaims.

1. An anticode oligomer complementary to bcl-2 mRNA consisting of from10-35 bases and comprising the nucleotide sequence TCTCCCAGCGTGCGCCAT(SEQ ID NO. 17).
 2. An anticode oligomer, wherein said anticode oligomeris an antisense oligonucleotide complementary to a portion of thepre-mRNA encoding the bcl-2 gene.
 3. The anticode oligomer of claim 2,wherein said anticode oligomer is an antisense oligonucleotidecomplementary to a portion of the region of the splice acceptor site orsplice donor site of the pre-mRNA encoding the bcl-2 gene.
 4. Ananticode oligomer, wherein said anticode oligomer is an antisenseoligonucleotide complementary to a portion of the 5′-untranslated regionof the bcl-2 mRNA.
 5. The anticode oligomer of claim 1, 2, 3 or 4,wherein said anticode oligomer contains at least one phosphorothioateand/or phosphoramidate modified nucleotide and is complementary to aportion of the pre-mRNA or mRNA encoding the bcl-2 gene.
 6. The anticodeoligomer of claim 5, wherein said anticode oligomer is aphosphodiester/phosphorothioate chimera.
 7. The anticode oligomer ofclaim 6 wherein the oligonucleotide comprises at least 2 to 3phosphorothioate linkages.
 8. The method of treating a bcl-2 relateddisorder comprising administering an effective amount of an anticodeoligomer, wherein said anticode oligomer hybridizes to the nucleic acidsequence of SEQ ID NO.
 19. 9. A method of treating cancer comprisingadministering an effective amount of an anticode oligomer, wherein saidanticode oligomer hybridizes to the nucleic acid sequence of SEQ ID NO.19.
 10. The method of claim 8 or 9, wherein said one or morechemotherapeutic agents are administered in combination with saidanticode oligomer.
 11. The method of claim 8 or 9, wherein saidcombination increases the sensitivity of said disorders tochemotherapeutic agents.
 12. The method of claim 8 or 9, wherein saiddisorder is selected from the group comprising non-Hodgkin's lymphoma,prostate cancer, breast cancer, gastro-intestinal cancer or coloncancer.
 13. The method of claim 8 or 9 for treating a human.
 14. Apharmaceutical composition comprising an amount of the anticode oligomerof any of claims 1-7 effective to prevent or inhibit a bcl-2 relateddisorder; and a pharmaceutically acceptable carrier.
 15. A method forincreasing the sensitivity of tumor cells to chemotherapeutic agents,comprising administering to the tumor cells an anticode oligomer,wherein said anticode oligomer hybridizes to the nucleic acid sequenceof SEQ ID NO.
 19. 16. The method of claim 15 wherein said cells expressthe human bcl-2 gene.
 17. A method of killing tumor cells, wherein saidcells express the human bcl-2 gene, comprising administering to thetumor cells one or more chemotherapeutic agents and an anticodeoligomer, wherein said anticode oligomer hybridizes to the nucleic acidsequence of SEQ ID NO.
 19. 18. The method of claim 17 wherein said cellsexpress the human bcl-2 gene.
 19. The method as in any of claims 15 to18, wherein said anticode oligomer hybridizes to the nucleic acidsequence TCTCCCAGCGTGCGCCAT (SEQ ID NO. 17).
 20. The method as in any ofclaims 15 to 18, wherein said chemotherapeutic agent comprises DTIC(decarbazine), Ara-C (cytosine arabinoside), MTX (methotrexate), taxol,cisplatin, etoposide, mitozantron, 2-chlorodeoxyadenosine,dexamethasone, mAMSA, hexamethyl melamine, mitroxantrone,antimetabolites, alkylating agents, plant alkaloids, antibiotics, andderivatives thereof.
 21. The method of claim 20 wherein saidantimetabolite comprises methotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, hydroxyurea, and 2-chlorodeoxy adenosine.
 22. Themethod of claim 20 wherein said alkylating agent comprisescyclophosphamide, melphalan, busulfan, cisplatin, paraplatin,chlorambucil, and nitrogen mustards.
 23. The method of claim 20 whereinsaid plant alkyloid comprises vincristine, vinblastine, and VP-6. 24.The method of claim 20 wherein said antibiotic comprises doxorubicin(adriamycin), daunorubicin, mitomycin c, and bleomycin.